In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions.Our Branch has generated a knock-in murine model for OI with a classical collagen mutation. A fundamental insight into OI mechanism derived from Brtl involves the role of ER stress, with elevated expressesion of CHOP, a member of the CREBP family activated by cell stress. At the level of proteomics, cell stress in Brtl skin was associated with differential expression of chaperones, metabolic enzymes and proteins related to cellular fate. More recently, Brtl osteoblasts have been shown to have impaired differentiation from stem cells to osteoblasts. The expression of early and late markers of osteoblast development was significantly reduced in Brtl. Conversely, Brtl MSCs generated more adipocytes than WT. Increased autophagy in Brtl osteoblasts suggests that ER stress contributes on impaired differentiation. Using Brtl, we completed a major therapeutic trial of bisphosphonate, which complements our pediatric trial. Alendronate treatment increased femoral DXA and cortical volumetric BMD. Brtl trabecular number and diaphyseal cortical thickness were improved, as was femoral stiffness and fracture load. However, detrimental changes were detected in material and cellular parameters of bone. Predicted material strength and elastic modulus of both Brtl and wild-type bone were deceased; brittleness of wild-type femora was increased. Furthermore, dramatic retention of mineralized cartilage may contribute to the weakening of bone material. Also, the function osteoblasts was impaired, with reductions in mineral apposition rate and bone formation rate. Osteoblast morphology was altered, making Brtl treated osteoblasts flattened. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. We have collaborated in a study demonstrating that a fluoresecent bisphosphonate analog is an accurate biomarker of deposition and retention in vivo. In addition, Brtl bone itself was demonstrated to have inherently increased susceptibility to microdamage using an ulnar loading model. This property makes OI bone more likely to become increasingly fragile from inhibition of microdamage repair by bisphosphonates. Brtl is currently being used for trials of potential pharmacological and non-traditional therapies. Much excitement in the osteoporosis research community focuses on the novel anabolic drugs that inhibit sclerostin. Treatment of Brtl with anti-sclerostin antibody led to improved bone mass and reduced femoral fragility without changes in tissue mineralization. Anti-sclerostin antibody represents a potential new therapy for pediatric OI patients. In a collaborative study, Brtl was used for an in utero cell transplantation trial of GFP expression stem cells. Despite low levels of engraftment, the perinatal lethality and femoral geometry and biomechanics of the engrafted Brtl mice were improved. The results are encouraging for translational trails. Second, we are modelling a lesson from type I OI to suppress mutant collagen expression. Specific suppression of transcripts of the mutant collagen allele can biochemically transform individual with severe OI into mild type I OI. We have introduced a Ribozyme target site into the BRTL mutant allele: we have generated transgenic mice expressing ribozymes targeted to the Brtl mutation. Preliminary data on Brtl/RZ mice is encouraging for improvement of Brtl biomechanical properties in female mice. We have identified a novel high bone density form of OI caused by mutations in the C-proteinase cleavage site of type I procollagen. The Asp-Ala dipeptide between the telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. We identified children with substitutions at two of these 4 peptides. They present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histomorphometry are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed, and in vitro cleavage by purified BMP1 is impaired. FTIR imaging of cortical and trabecular bone confirms elevated mineral/matrix ratios in affected children, compared to normal controls and classical OI samples, as well as significantly increased collagen maturity in trabecular bone BBD (Bone mineralization density distribution) reveals a marked shift toward increased mineralization compared to controls. The data not only reveal a novel form of OI but also provide new fundamental insight on roles of procollagen processing and the mechanism of tissue mineralization. We are currently generating a murine model for high bone density OI, in order to study the molecular and biochemical mechanism of the mineralization, and its developmental progression. To better understand the relationship of genotype and phenotype in human OI, the BEMB led an international consortium of connective tissue laboratories to assemble and analyze a mutation database. The initial database published in 2007 contained over 830 mutations; currently the database under analysis contains over 1300 mutations. Genotype-phenotype modeling revealed different functional relationships for each chain of type I collagen. Lethal mutations in alpha 1 (I) coincide with the Major Ligand Binding Regions. Lethal regions in alpha 2(I) continue to support the Regional Model first proposed by the BEMB, with lethal mutations in regularly-spaced clusters along the chain that coincide with proteoglycan binding regions. This model correctly predicts clinical outcome in 86% of alpha 2(I) mutations. We are also continuing our clinical studies of children with types III and IV OI. The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The treatment group experienced improvement in vertebral parameters, which leveled off after one to two years of treatment. There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Hence the changes previously reported appear to have been a placebo effect in uncontrolled trials. We are recommending that treatment of children with types III and IV OI with pamidronate be limited to at most three years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial. We are also focusing on the variability of response to treatment in each group. The improvements in vertebral height and area do not correlate with changes in DXA z-score, nor did the improvement in vertebral height and area correlate for individual children. These differences highlight the inadequacy of DXA as a surrogate for bone strength. We have also focused on the cardiopulmonary aspects of OI. Findings in the NIH pediatric OI cohort revealed a significant progressive decline in pulmonary function and increased restrictive pulmonary disease independent of scoliosis. Most participants in the study showed mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. This data will change the standard of anticipatory care for OI and promote early detection and treatment of the greatest cause of mortality in OI.